DE2105009C3 - Processes for the cleavage of racemi see amino acids in the optically active components - Google Patents

Description

45

The invention relates to the optical resolution of racemic amino acids and in particular to the asymmetric resolution Hydrolysis of u-N-acyl-nL-amino acid and the subsequent recovery of the optically active components.

It is known that molds such as Aspergillus oryzae produce acylase. This mold acylase has the ability to hydrolyze the 1st isomer of acyl amino acid to the free amino acid. Furthermore, it is also known that the n-N-acyl-di.-amino acid Can be hydrolyzed asymmetrically by common acylase, whereby this Raccmai easily into the optically active enantiomers !! can be split. After the hydrolysis is complete the reaction solution is boiled and or acidified to precipitate the enzyme, and then the Enzyme precipitate filtered off. In this way, Schimmelpil / acvlase can only be used once. us and it must then be discarded. Hence lies the wasteful use of the acylasc On the hand. In addition, the recovery of manufactured amino acid necessarily requires one additional step to remove the enzyme and its contaminant products from the final product. In addition, mold acylase in an aqueous solution is against environmental factors sensitive, which can reduce their activity relatively quickly even when the enzyme reaction is carried out at the optimal temperature.

L'm to overcome the disadvantages of the previously mentioned working methods listed above different methods used. For example, U.S. Patent 3,243,356, Canadian Patent 659 059 and USA patent 3 3S6 888 ι ■ optical cleavage of DL-amino acid described by asymmetric hydrolysis of their N acyl derivative with water-insoluble enzyme preparations. The water-insoluble polypeptidyl derivatives of Mold acylase. described in U.S. Patent 3,243,356 are made by copolymerization produced by acylase with anhydrides of certain «-N-carboxyamino acids. The enzyme preparation, ". oak is described in the Canadian patent shrill. is an insoluble, porous plastic, in which acylase from kidney is embedded. the U.S. Patent 3,386,588 method involves incorporating mold acylase into an anion exchange polysaccharide adsorbent to form a practically insoluble complex and supply a solution of an N-acyl derivative from Raccmischer Amino acid to the acylase complex. In the first and second modes of operation, i. H. USA patent 3,243,356 and Canadian Patent 659,059, however, it is a disadvantage that the two Acylase preparations of these methods do not have high enzymatic activity due to a noticeable loss able to ensure the activity in the course of their production, since the crosslinking method U.S. Patent 3,243,356 as well as the "inclusion method" of Canadian Patent 659 059 complicated procedures including demanding treatment conditions require, thereby causing the enzyme to break down. In the third method, i. H. USA patent 3,386,888, the acylase tends to be released from the enzyme-adsorbent complex when the concentration of a substrate is set higher than about 0.3 M, so that the production from optisc !; active amino acid can be accomplished within a short period of time.

It is the object of the invention. a new, water-insoluble one Manufacture mold acylase preparation, which has high enzymatic activity during for a long period of time and the acylase does not in the presence of a high substrate concentration releases. Another object of the invention is to provide an improved method for the optical splitting of to deliver racemic amino acids by using acylase, which the need of the Avoids rejection of the enzyme and on the contrary allows it to be reused in a number of consecutive operations. Farther It is the object of the invention to provide a method that eliminates the need for additional Steps for separating the desired product from others present in the reaction mixture Avoid substances, in particular from the enzyme itself, and in the case of the optically active i.-isomer in hi'lien yields is produced.

The process according to the invention is characterized in that mold acylase is treated with a practically water-insoluble haloetylpolysacchaid, a solution of an alkyl derivative of the racemic amino acid is added to the acylase preparation obtained and the solution obtained is incubated according to methods known per se.

Bromoacetyl cellulose, bromoacetyl dextran. Bromine- ! .ciylagarose *). Chloroacetyl cellulose. Chloraeetvileuran. Chloroacetyl agarose. Iodoacetyl cellulose. Jodi Ethyldextran and iodoacetyl agarose are suitable as a water-insoluble haloacetyipohviccharide of the invention. "These poly -. echaride derivatives can according to the US patent 3,278,392 method described. For example, a mixture of (Allulose powder. Bromoacetic acid and dioxane at 20 t-.i-. Stirred at 30 ° C. for 16 to 24 hours. To the mixture v. bromacetyl bromide is added. After stirring for 6 to 8 hours, the mixture obtained is in 1: 1 · poured water, and the precipitates formed are washed and dried, whereby Bromaceiyl cellulose powder is obtained in the pure state. The other bromoacetyl polysaccharides and the (Chloracetyl polysaccharides are obtained in the same manner as above. The content of chlorine or Bromine in the polysaccharide derivatives !! can easily by adjusting the amount d.; Halogenacety! halides used in the above reaction be managed. Alternatively, iodineetylpolviccharide can be used by adding the relevant bromoacetyl or chloroacetyl polysaccharide to a 95% strength Alkanol solution of sodium iodide, followed by agitation of the resulting mixture at room temperature can be made over one night. The haloacetyl polysaccharide. which 4 to Contains 40 wt / wt percent of a halogen atom, especially the polysaccharide derivative. soft about 8 weight percent chlorine, about 18 weight percent bromine, or about 31 weight percent Contains iodine is applicable for purposes of the invention.

The water-insoluble one to be used in the invention Acylase preparation can be made by using any of these haloacetylpolysacharides Mold acylase in a solvent, preferably in the presence of a salting out agent for protein. z. B. ammonium sulfate. Sodium sulfate, in contact is brought. When a haloacetyl polysaccharide. which the above concentration on one Halogen is used, is preferred Amount of mold acylase. which is to be introduced into the polysaccharide, about 0.1 to 0.25 g / g haloacetyl polysaccharide. The originated. water-insoluble acylase separation is determined by filtration separated or centrifuged, and optionally the preparation can be further by washing with cleansed with a physiological saline solution. It is preferred as a source for the aylase enzyme preparations to use which consists of microorganisms, e.g. B. Aspergillus ory / .ae. Aspcrgillus meleus, Aspergillus midulans, Penicillium vinaceum or Penicillium corymbiferum. obtained aylase contain. You can use the methods given above in the preparation of the water-insoluble enzyme preparation

*) Agarose = neutral galakiosis polymer. soft ·, for the Gclfesligkcil of agar-agar isl.

Procedures applied after dissolution in water will. Watery. Aeylase-containing solution can be obtained directly by culturing one of the above Microorganisms are produced in an aqueous medium and filtering off the resulting culture. In addition, the solution can also by inoculating one of the microorganisms mentioned a wheat bran hull medium which has previously been treated in an autoclave. Incubate the mixture

ίο under suitable conditions and extracting the resulting culture can be made with water. Alternatively, acylase solution can be used. what different Contains impurities such as protein impurities and coloring matter, as such Impurities easily from the water-insoluble enzyme prepuraiion by subsequent washing with Water can be removed.

Many N-acyl derivatives of racemic amino acids are used as substrates in the present invention or starting material used. Examples of these N-acylamino acids are as follows: a-N-acetylamino acids like N-Acety! alanine. N-acetylvaline. N-acetylleucine, N-acetylisoleucine. N-acetylserine. N-acetylthreonine. N-AcUv Icy stone. N-acetylmethionine.N-acetylphenylalanine. N-acetyl tyrosine. N-acet \! Aspartic acid. N-Aeely! Glutamic acid. N-acetyl histidine. fi-N-j-N-diac? lyllysine. n-N-i-N-diacetylornitine and K-N-acetyl-f-N -benzoyl lysine. Other acyl derivatives like N-formyl-. N-chloraceiyl-. N-bromoacetyl-, N-propionyl-. N-butryl or N-benzoyl derivatives of α-amino acids are also used.

The concentration of such applicable Substrate is not critical for purposes of the present invention. In a preferred embodiment of the invention is the racmate of an n-N-acyl amino acid but dissolved in water at a concentration of 0.2 to 1.0 mol 1. The solution will then adjusted to a pH of 5.0 to 9.0. The aforementioned, water-insoluble enzyme preparation is added to the solution, and the mixture is taken at a temperature of 30 to 60 C Stir incubated for sufficient time to complete the reaction. It's beneficial a small amount of cobalt (II) ions to the substrate solution to be added as an activator. After the completion of the reaction, the mixture is filtered or centrifuged to recover the insoluble enzyme for subsequent use. The 1.-isomers, which is in the form of free amino acids, can be obtained from the filtrate or the mother liquor. Alternatively, the enzymatic Hydrolysis according to the invention can be carried out by a column method, which makes it possible is. to carry out the hydrolysis sequentially.

For example, the water-insoluble acylase preparation is loaded onto a column, and an aqueous one Solution (pH 5.0 to 9.0) of a-N-acyl-UL-amic acid is passed through the column at a suitable flow rate, creating an aqueous Solution, the 1st amino acid and «-N-acyl-D-amino acid contains, is obtained as an eluate. From the eluate the proportion of the amino acid present in the free form and the proportion of that in the acylated form existing amino acids from each other with the aid

f> 5 would assume the difference in their solubilities Water or an organic solvent. In any case, the water-insoluble retains Acylase preparation of the invention the high values

the enzymatic activity during the reaction even in the presence of a high concentration of a Substrates at, e.g. B. in a solution which has a concentration of 0.3 to 1.0 Moll of substrate contains. In addition, as a result of the sufficient stability of the enzymatic activity of the enzyme preparation According to the invention, the repeated use of the acylase preparation is also possible.

In the examples, the activity of each of the enzyme preparations is given in the form of the unit, such as to it is described in "Report of the Commission on Enzymes of the International Union of Biochemistry (1961)" so that the acuity represents the number of micromoles of the substrate in units, which was hydrolyzed by the action of the enzyme in 1 minute if after any of the using the following methods:

Free enzyme

The substrate solution contains: 0.2 M solution of N-acetyl-DL-methionine (pH = 7.0) 0.5 ml

1.5 x 10 * ³ M solution of CoCl ₂ 0.5 ml

0.1 M phosphate buffer solution
(pH = 7.0) 1.0 ml

To this solution, 1.0 ml of the acylase solution is added and the mixture is allowed to stand for 30 minutes incubated at 37 "C. The amount of L-methionine released is determined colorimetrically using the ninhydrin method examined.

Insoluble enzyme

The substrate solution contains acetyl-DL-methionine in a concentration of 0.2 M and CoCl ₂ in a concentration of 5 × 10 " ⁴ M (pH = 7.0). The substrate solution is passed through a column filled with the insoluble enzyme at a Space velocities of 5 and 37 ° C passed, l methionine in the eluate is determined in the same manner as described above.

The invention is explained in more detail below with the aid of examples, without restricting it.

B e i s ρ i e 1 1

A solid culture, which was herger.lellt by cultivating Aspergillus ory / ae on a medium from wheat clump shells, is extracted with ten times the volume of water with stirring for 2 hours. Gradually ammonium sulfate to d ^ m; until the ammonium sulfate concentration reaches o extract added, the saturation of 0.3 times in your extract, and the resulting precipitates are removed by centrifugation. Further ammonium sulfate is added to the mother liquor or supernatant liquid until the concentration thereof reaches 0.5 times saturation in the solution. The precipitates thus obtained are collected by centrifugation and dissolved in water. The solution is dialyzed against running water and then lyophilized. The acylase activity of the lyophilized enzyme is 0.4 units / mg.

100 mg of the enzyme are in 50 ml of a 0.2 M Phosphorus buffer solution (pH = 8.5) dissolved. 10 g ammonium sulfate and 1 g of bromoacetyl cellulose containing 18.1 weight percent bromine becomes added to the solution. The mixture is stirred for 24 hours at 7 ° C, and the insolubles are collected by filtration, thereby obtaining 2.5 ml of a water-insoluble enzyme preparation will. The acylase activity is 48 U / ml.

A mixture of 2.5 ml of the enzyme preparation and 2.5 ml of cellulose powder is filled into a column of 1 × 6.3 cm. An aqueous solution (pH = 7.0) containing a concentration of 0.2 M N-acetyl-Tu-methionine and a concentration of 5 x 10 ~ ⁴ M cobalt ions is continuously through the column at 37 ° C and flow rates of 40 and 20 ml / hour sent through. The concentration of L-methionine in the elual is determined colorimetrically by the ninhydrin method on samples obtained at intervals, and the conversion rate of N-acetyl-DL-methionine to L-methionine is calculated therefrom. The results obtained are shown in Table 1.

Tabeiie I

Conversion rates of N-acetyl-DL-melhionin
to L-methionine (%)

3 treatment time Flow velocity 20ml hour (Hours.) 40 ml hours 100 7th 68.3 100 21 68.6 100 48 68.3 100 75 68.5 100 96 68.6 100 120 68.5 Example 2

100 mg of the same, purified enzyme, as used in Example 1, are in 50 ml of 0.2 M Dissolved phosphate buffer solution (pH = 8.5). 10 g ammonium sulfal and 1 g of chloroacetyl cellulose. which contains 8 percent by weight of chlorine, become the Solution added. Then the mixture is treated in the same manner as in Example 1, wherein 2.5 ml of a water-insoluble enzyme preparation with an acylase activity of 25 U / ml can be obtained.

A mixture of 2.5 ml of the enzyme preparation and 2.5 ml of cellulose powder is filled into a column of 1 × 6.3 cm. An aqueous solution (pH = 7.0), which contains a concentration of 0.2 M N-acetyl-DL-methionine and a concentration of 5 × 10 " ⁴ M cobalt ions, is continuously passed through the column at 37" C and flow rates of 25 and 12.5 ml / hour passed through. The concentration of L-methionine in the eluate is determined from samples obtained at time intervals, and the conversion rate of N-acetyl-DL-methionine to L-methionine is calculated therefrom. The results obtained are shown in Table II.

Table Π

Conversion rates of N-acetyl-di.-methionine
to L-methionine (%)

Treatment time
(SId.)

2
21

Speed

25 ml / hour

69.1
69.3

12.5 ml hours

100
100

Trading time
(Sid.)

48

75

96

120

continuation 12.5 ml / sld 100 Flow rate 100 25 ml / Sld. 100 68.9 100 69.0 69.3 68.8

Example 3

100 mg of the same, purified enzyme, as used in Example 1, are "in 50 ml of 0.2 M Phosphate buffer solution (pH = 8.5), dissolved. IO g of ammonium sulfate and 1 g of iodoacetyl cellulose, which 30.6 percent by weight iodine is added to the solution. Then the mixture is in treated in the same way as in Example 1, whereby 2.5 ml of a water-insoluble enzyme preparation with an acylase activity of 80 U / ml.

A mixture of 2.5 ml of the enzyme preparation and 2.5 ml of cellulose powder is poured into a column measuring 1 × 6.3 cm . An aqueous solution (pH = 7.0). having a concentration of 0.2 M N-acetyl-Tu-methionine and contains a concentration of 5 x 10 ^-4 M of cobalt ions is continuously through the column at 37 "C and flow rates of 40 and 20 mi / h. du The concentration of i.-methionine in the eluate is determined from samples obtained in time intervals, and the conversion rate from N-acetyl-nL-methionine to L-methionine is calculated from this. The results obtained are shown in Table III listed.

Table III

Conversion rates of N-acetyl-iM.-methionine
to i.-methionine (%)

3chandhings / .eil 1 license speed wedge iel 4 20 ml hours (SId.) 40 ml hours 100 1 67.6 100 21 67.7 100 48 67.3 100 75 67.8 100 96 67.4 100 120 67.7 Ex

A mixture of 0.5 ml of the same enzyme preparation as used in Example I, and 4.5 ml of cellulose powder was filled into a column 1 × 6.3 cm. An aqueous solution (pH = 7.0), which contains N-acetyl-DL-methionine and a concentration of 5 · 10 ~ * M cobalt ions passed through the column at 37 or 52 C at flow rates of 25 or 12.5 ml hr. the Conversion rate of L-methionine is calculated in each case. The results obtained are in the Table IV shown.

Alternatively, the procedure described above was repeated, with the exception that the same enzyme preparation as used in Examples 2 or 3 in place of the above Dissection was used and included in each

Results obtained in these cases are shown in Tables V and VI. Table V gives the results obtained using the enzyme preparation of Example 2 and Table VI the results obtained using the enzyme preparation of Example 3 again.

Table IV

{onzcnlration of Flow N-acctyl fast- ni.-mcthionine kcil (Minor) (ml / h) 0.2 25th 0.2 25th 0.5 12.5 0.5 12.5

Temperature IC)

37 52 37

52

Conversion rod to i.-methionine

36.6 61.5 31.5 59.4

Table V

Concentration of

N-acetyl

Di-methionine

(Minor)

0.2
0.2
0.5
0.5

Flow velocity
speed
(ml hours)

25th
25th
12.5
12.5

Temperature (C)

37 52 37 52

Conversion rate to ι -methionine

19.3 31.5 16.9 30.2

Table VI

concentration of Flow temperature Conversion N-acctyl swiftly advise i> L-methionine speed ( ⁷ C) L-methionine (Minor) (ml / h) 37 (%) 0.2 25th 52 75.2 0.2 25th 37 100 0.5 12.5 52 68.4 0.5 12.5 100

Example 5

An aqueous solution containing amino acid N-Acrtyl nL and contains a concentration of 5 x 10 ^-4 M of cobalt ions is adjusted to a pH of 7.0. The solution is passed through the same column as used in Example 1 at 37 ^° C and a flow rate as shown in Table VII. The conversion rate of N-acetyl-dl-amino acid to L-amino acid is calculated in the same way as described in the previous examples. The results obtained are shown in Table VII .

Alternatively, the above procedure was repeated, but with the exception that the same Column as used in Examples 2 or 3 in place of the above column, and the i The results obtained in each of these cases are shown in Tables VIII and IX, with Table VU the results obtained using the column of Example 2, and Table IX that of Ve application of the column of Example 3 results obtained Ergel reproduces.

309 646/3

Table VII

N-Acety] -i> l.-amino acid

N-acetyl-DL-methionine ..
N-acetyl-DL-methionine ..

N-acetyl-DL-valine

N-acetyl-DL-phenylalanine
N-acetyl-ni.-tryptophan.

Concentration (mol / l)

0.2 0.5 0.2 • 0.2 0.2

Conversion ring to! - amino acid ("/,)

Flow speed (ml / sld.)

formed i.-amino acids

L-methionine

L-meth.ionine

L-valine L-phenylalanine

L-tryptophan

40

68.8 100 52.5 89.8 53.5 87.2 68.5 100 56.8 93.0

12.5

100
100

100

Table VIII

N-acetyl-ni.-amino acid c

N-acetyl-DL-methionine ..
N-Acelyl-nL-methionine ..

N-acetyl-DL-valine

N-Acctyl-DL-phenylalanine
N-acetyl-DL-tryptophan.

Concentration (mol / l)

0.2 0.5 0.2 0.2 0.2

formed! .- amino acid

Conversion rates to [.-Amino acid (%)

Flight speeds (ml / hour)

20 I 10 I 5

L-methionine

L-methionine

L-valine

L-phenylalanine

L-tryptophan

69.2 100 53.6 89.9 56.7 91.2 69.4 100 60.4 98.4

100
100

100

N-acctyl-ui.-amino acid c

N-acetyl-DL-methionine ..
N-acetyl-DL-methionine ..

N-acetyl-DL-valine

N-acetyl-DL-phenylalanine
N-acetyl-DL-tryptophan.

Table IX

Formed! .- Amino acid conversion rates to! .- Amino acid (%)

Flow rates (ml / hour)
80

L-methionine

L-methionine

L-valine

L-phenylalanine

L-tryptophan 68.7
50.4
59.7
68.4
60.7

40 20th 100 67.9 100 89.4 100 100 - 98.3 100

Example 6

114.7 g of N-acetyl-DL-methionine are poured into 300 ml 2N sodium hydroxide dissolved. 357 mg cobalt chloride hexahydrate are added to the solution and the solution is diluted to a total volume of 3 liters with water. The solution will be through the same column as used in Example 1 at a flow rate of 20 ml / hour sent through at 37 ° C. The eluate is reduced to about one fifth under reduced pressure its original volume concentrated, whereby the greater part of the L-methionine crystallizes out will. The crystals are collected by filtration and the filtrate is evaporated to dryness. 50 ml of ethanol are added to the residue, and the remaining crystals (L-methionine) are collected by filtration. Both crystal fractions are combined with one another and recrystallized from aqueous ethanol to give 40.8 g of L-methionine; [α]? = + 23.4 ° (c = 3, in 1 N-HCl).

The ethanol fraction is evaporated to dryness. The residue obtained in this way is dissolved in 100 ml of water. The aqueous solution is passed through a strongly acidic cation exchange resin column, and then the column is washed with water. The eluate and the washing liquids are combined with one another and concentrated to dryness. The residue is recrystallized from water, 50.6 g of N-acetyl-D-methionine being obtained; Mp = 103-104 ° C; [a] ' _o ⁵ = -19.6 ° (c = 3 in water).

9.6 g of this compound are in 85 ml of 2 N salts acid refluxed for 2 hours. The reaction mixture is evaporated to dryness. Dc The residue is adjusted to pH = 5.5 with 2N sodium hydroxide and extracted from aqueous ethanol recrystallized to give 5.3 g of D-methionine; [α]? = 23.4 ° (c = 3, in 1N HCl).

Example 7

82.9 g of N-acetyl-DL-phenylalanine are dissolved in 200 m 2 of N sodium hydroxide. 238 mg cobalt chlo Ride hexahydrate are added to the solution and the solution is diluted with water volume of 21 diluted. The solution is through the same column as used in Example 1 was at a flow rate of 15 ml / hour. sent through at 37 ° C The eluate win concentrated under reduced pressure to about one fifth of its original volume. Dl

precipitating crystals are collected by filtration, and the filtrate is evaporated to dryness. 40 ml of ethanol are added to the residue and the remaining crystals (1-phenylalanine) are collected by filtration. Both crystal fractions are combined with one another and recrystallized from water, 29.6 g of L-phenylalanine being obtained; [ä] " = -34.4 ° (c = 1, in water).

The ethanol fraction is evaporated to dryness and the residue is crystallized from dilute hydrochloric acid. The crystals which precipitate are recrystallized from water, 37.4 g of N-acetyl-D-phenylalanine being obtained; Mp. 170 ^° C; [„]» = -50.5 ° (c = 1, in absolute ethanol).

10.4 g of this compound are refluxed in 85 ml of 2N hydrochloric acid for 2 hours. The reaction mixture is evaporated to dryness. The residue is adjusted to pH = 5.5 with 2N sodium hydroxide and recrystallized from aqueous ethanol, 6.4 g of D-phenylalanine being obtained; [α] ?, ⁷ = + 34.9 ° (c = 1, in water).

Example 8

8.29 g of N-acetyl-DL-phenylalanine are in 20 ml Dissolved 2 N sodium hydroxide. 24 mg cobalt chloride hexahydrate are added to the solution, and the solution is diluted with water to a total volume of 200 ml. 2.5 ml of the same Enzyme preparation as used in Example 1 are added to the solution. The The mixture is incubated at 37 ° C. for 24 hours with stirring. Then the mixture is in the same Treated manner as in Example 5, giving 2.72 g of L-phenylalanine and 3.52 g of N-acetyl-D-phenylalanine can be obtained. In the course of the reaction, the rate of conversion of L-phenylalanine is measured.

Alternatively, the above procedure is repeated, but with the exception that the same Enzyme preparation used in Example 2 or 3 is used, and in each of these cases the rate of conversion to L-phenylalanine is measured in the course of the reaction. The results obtained are shown in Table X.

Table X Conversion Rates to L-Phenylalanine (%)

Enzyme preparation

In example 1
applied preparation
In example 2
applied preparation
In example 3
applied preparation

Incubation period (Sld.)

39.4

40.5

64.5

7th G 12th 14th 24 48 61.8 - - - 100 - - 42.5 - 70.2 - 100 - - 100 - - -

Claims (6)

Patent claims: 1. Process for the resolution of racemic amino acids into the optically active components. d a d u r c h e k e η η ζ e i c h η e t that mold acylase is treated with a practically water-insoluble Halogep.acetylpolysaccharid, a Solution of a u-N-acyl derivative of the racemic Amino acid is added to the acylase preparation obtained and the resulting solution according to known methods is incubated. 2. The method according to claim 1, characterized in that that the solution flow practically continuously in contact with the enzyme preparation is left. 3. The method according to claim 2, characterized in that that the solution of a / i-N-acyl derivative of the racemic amino acid in an aqueous Medium is allowed to flow continuously through a column of the enzyme preparation and the resulting, free i.-amino acid and the d-N-acyine amino acid separated from each other in the eluate. 4. The method according to claim 1, characterized in that that as the haloetyl polysaccharide bromoacetyl cellulose. Bromoacetyldextran. Broin Acetyl Agarose. Chloroacetyl cellulose. Chloroacetyl xtran. Chloroacetyl agarose. Iodoacetyl cellulose. Iodoacetyldextran or Iodoacetylagarose is used. 5. The method according to claim 1, characterized in that that as a mold acylase from Aspergillus oryzae or Penicillium vinaceum obtained acylases can be used. 6. The method according to claim 1, characterized in that that a practically water-insoluble mold acylase preparation is used, which by treating mold acylase with a practically water-insoluble halogen acetyl polysaccharide in the presence of a salting out agent for protein.